Multimeric fusion proteins of TNF superfamily ligands

ABSTRACT

A method for constructing stable bioactive fusion proteins of the difficult to express turn or necrosis factor superfamily (TNFSF), and particularly members CD40L (CD 154) and RANKL/TRANCE, with collectins, particularly pulmonary surfactant protein D (SPD) is described. Single trimers of these proteins lack the full stimulatory efficacy of the natural membrane forms of these proteins in many cases. The multimeric nature of these soluble fusion proteins enables them to engage multiple receptors on the responding cells, thereby, mimicking the effects of the membrane forms of these ligands. For CD40L-SPD, the resulting protein stimulates B cells, macrophages, and dendritic cells, indicating its potential usefulness as a vaccine adjuvant. The large size of these fusion proteins makes them less likely to diffuse into the circulation, thereby limiting their potential systemic toxicity. This property may be especially useful when these proteins are injected locally as a vaccine adjuvant or tumor immunotherapy agent to prevent them from diffusing away. In addition, these and other TNFSF-collectin fusion proteins present new possibilities for the expression of highly active, multimeric, soluble TNFSF members.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for preparing solublemultimeric proteins consisting of more than three iterations of the samebioactive molecule using recombinant DNA technology.

The present invention particularly concerns a new method of producingmultimeric fusion proteins involving the TNF superfamily (TNFSF) membersas fusion proteins with SPD, and more specifically, CD40L-SPD fusionproteins and useful modifications thereof.

2. Description of Related Art

Numerous proteins can be made using modern molecular biology techniquesand used in diagnostic and therapeutic applications. Using recombinantDNA techniques, the DNA encoding a single amino acid chain isconstructed and then introduced into a cell which manufactures the finalprotein. Some cells, especially bacteria like E. Coli, lack the abilityto properly fold the amino acid chains into the proper quaternarystructure and they often fail to apply the necessary modifications(e.g., glycosylation and disulfide bond formation) that are needed forthe protein to be bioactive and resistant to degradation in vivo. Whilemost of these challenges can be met by expressing the amino acid chainin eukaryotic cells like yeast or mammalian cells in vitro, it is notalways straightforward to express proteins that consist of two or moreamino acid chains. In general, for multichain proteins, the single aminoacid chains must associate together in some way either within theproducer cell or subsequently after the monomers are secreted from theproducer cell. For artificially constructed molecules, the introductioninto a single amino acid chain of an amino acid sequence which causesthis chain-to-chain association can be an important step in producingmultichain proteins.

One of the most widely used methods of causing two amino acid chains toassociate is to conjoin, at the DNA coding level, segments from theprotein of interest and a segment from a spontaneously dimerizingprotein. The best example is to conjoin or fuse a protein with the Fcportion of immunoglobulin, creating a dimeric Fc fusion protein (Fanslowet al., J. Immunol. 136: 4099, 1986). A protein of this type can beformed from the extracellular domain of a tumor necrosis factor receptorfused to Fc (termed etanercept and marketed as ENBREL®), which iseffective in the treatment of rheumatoid arthritis. A second example isthe construction of a fusion protein between the dimerizingextracellular portion of CD8 with the extracellular portion of CD40L(Hollenbaugh et al., EMBO J. 11: 4313, 1992). Here, the dimerizing CD8portion of the fusion protein helps to maintain the CD40L portion in thetrimeric form needed for its bioactivity. A more recent example is theaddition of an isoleucine zipper motif to CD40L, which permits theproduction of trimeric soluble CD40L molecules (Morris et al., J. Biol.Chem. 274: 418, 1999).

The TNF superfamily (TNFSF) consists of an expanding number of proteins(see Table I) which are crucial for the development and functioning ofthe immune, hematological, and skeletal systems. TNFSF proteins areligands for a corresponding set of receptors of the TNF receptorsuperfamily (TNFRSF). All TNFSF members are expressed as Type IImembrane proteins, with the exception of lymphotoxin-alpha which isproduced as a secreted protein. However, soluble forms of several TNFSFproteins can be released from the cell surface by proteolytic cleavage,usually by specific metalloproteinases.

The production of soluble forms of TNFSF proteins has been an importantstep in the study of these proteins. Soluble TNFSF ligands can be usedto study the activities of these proteins in vitro without thecomplexities in interpretation that result when cells or cellularmembranes expressing TNFSF proteins are added. In addition, solubleforms of several TNFSF proteins have potential as therapeutic agents forhuman diseases. In particular, TNF-? has been extensively studied forthe treatment of cancer and soluble CD40L is currently undergoingclinical trials to assess its antitumor effects.

To produce soluble forms of TNFSF proteins, either the membrane proteinis expressed in a cell line possessing a protease capable of separatingthe TNFSF extracellular domain from the transmembrane domain or atruncated form of the TNFSF protein is produced which consists solely ofthe extracellular domain plus a signal sequence. In either case, certainsoluble forms of TNFSF proteins are unstable in solution as simplehomotrimers composed solely of the extracellular domain. For example,naturally solubilized TNF-? is labile under physiological conditions[Schuchmann, 1995 #129]. To solve this stability problem, chimericproteins have been constructed according to one of four different designprinciples: (1) The extracellular portion of the TNFSF protein has beenexpressed fused to the dimeric portion of the immunoglobulin Fc fragmentU.S. Pat. No. 5,155,027, Oct. 13, 1992, issued to, Andrzej Z.Sledziewski, et al. In the case of CD40L and OX40L, this yields asoluble molecule which is significantly less active than the nativemembrane form of this protein. (2) The extracellular portion of theTNFSF protein has been expressed with an antigenic tag (usually the FLAGmotif) fused to its N-terminus [Mariani, 1996]. The addition of anantibody to the tag (e.g., anti-FLAG antibody) aggregates these proteinsinto a multimeric form. Crosslinking enhances activity on B cells. (3)The extracellular portion of the TNFSF protein has been expressed fusedto the spontaneously dimerizing extracellular portion of the CD8molecule [Hollenbaugh, 1992]. In the case of CD40L, this creates ahexameric molecule [Pullen, 1999] which is likely formed by two CD40Ltrimers attached to three CD8 dimeric stalks. Despite this, the additionof an anti-CD8 antibody to crosslink the CD40L-CD8 fusion protein yieldsa further enhancement of CD40L activity on B cells. (4) Theextracellular portion of the TNFSF protein has been expressed fused to atrimerizing isoleucine zipper which maintains the overall trimericstructure of the protein [US Pat. No. 5,716,805, Feb. 10, 1998, issuedto Subashini Srinivasan et al. This soluble CD40L trimer or ‘sCD40LT’ isthe form of that protein now being clinically tested in humans for itsanti-tumor effects.

Compounding the difficulties in producing stable forms of soluble TNFSFproteins are compromises in bioactivity. As exemplified by FasL, TNF,and CD40L, many of the soluble forms of these proteins lack the fullrange of stimulatory activities displayed by the membrane forms of thesemolecules. For FasL, several groups have reported that naturallyproduced soluble FasL (generated by proteolytic cleavage from themembrane form) has a spectrum of activities that is distinctly differentfrom the membrane form. Soluble FasL induces apoptosis in activated CD4+T cells but not fresh, resting CD4+ T cells. In contrast, both types ofCD4+ T cells are killed by membrane FasL or a recombinant soluble formof FasL (WX 1) that spontaneously aggregates into oligomers larger thana decamer. For TNF, T cell activation through stimulation of TNFR II,the 80 kDa receptor for TNF, is much greater with membrane TNF′ thansoluble TNF. However, if soluble TNF is produced as a tagged protein andcrosslinked with an antibody against the tag, then it completely mimicsthe activities of membrane TNF [Schneider, 1998]. Finally, for CD40L,the stimulatory effects of a soluble form of this TNFSF protein areenhanced by crosslinking [Kehry, 1994] and yields an activity similar tomembrane CD40L. For example, soluble CD40L-CD8 fusion protein requirescrosslinking with a antibody to CD8 in order to drive resting B cells toproliferate to a degree similar to membrane-bound CD40L.). Even morestrikingly, although membrane-bound CD40L expressed onbaculovirus-transduced SF9 insect cells is a strong B cell stimulus,small vesicles (10-1,500 nm) prepared from the membranes of these cellsare less stimulatory. However, ultracentrifugation of these vesiclescreates aggregates which have the full activity of the original membraneCD40L protein. This indicates that B cells are more highly stimulated bya large surface of CD40L than they are by a smaller surface expressingthis membrane ligand.

Taken together, the above reports suggest that, for some TNFSF/TNFRSFligand/receptor pairs at least, it is essential to cluster receptorstogether for full signaling activity. By this interpretation, theefficacy of the membrane forms of FasL, TNF, and CD40L occurs becausethese ligands can move in the plane of the membrane toward the contactzone with a receptor-bearing responding cell, thereby clustering ligatedreceptors to form a receptor-dense region of the membrane. Thisinterpretation is further supported by experiments where crosslinking ofa soluble TNFSF protein effectively mimics the activity of the membraneform of the protein [Scheider, 1998].

In all of the above examples, no more than three amino acid chains havebeen caused to associate together. There is a need to produce multimericprotein molecules where more than three amino acid chains are caused toassociate into a single soluble molecular complex. An important examplecomes from studies of CD40L (also called CD 154 or TNFSF5), which is amember of the TNF family of molecules that are normally expressed asinsoluble, cell membrane proteins. It has been shown that solublehomotrimers composed of the extracellular regions of CD40L, TNF, andFasL are not potently active on resting cells that bear receptors forthese proteins. However, if these proteins are expressed with a tag ontheir ends (e.g., the FLAG psptide sequence) and then the trimers areextensively crosslinked using an antibody to FLAG, full activity appears(Schneider et al., J. Exp. Med. 187: 1205, 1998). From this, it can beinferred that the soluble single-trimer forms of these molecules doesnot duplicate the multivalent interactions that normally occur when areceptor-bearing cell comes in contact with the membrane of a cellexpressing numerous ligand trimers on its surface. This distinction maybe due to a need for receptor clustering for full signaling (Bazzoni andBeutler, N. Engl. J. Med. 334: 1717, 1996), which in turn is onlypossible with a multimeric ligand engaging many receptors at the sametime in a localized region of the cell membrane.

SUMMARY OF THE INVENTION

The present invention contemplates a method of preparing soluble,multimeric mammalian proteins by culturing a host cell transformed ortransfected with an expression vector encoding a fusion proteincomprising the hub, body, and neck region of a collectin molecule and aheterologous mammalian protein.

In one embodiment, the heterologous mammalian protein comprises an extracellular domain of a mammalian transmembrane protein; the resultingfusion protein forms a multimer.

In another embodiment, the heterologous mammalian protein comprises asoluble protein such as a cytokine; the resulting fusion protein forms amultimer.

In another embodiment, sites of proteolytic degradation are included orremoved from the fusion protein; the resulting fusion protein forms amultimer from which are cleaved single units at a rate made variable bythe nature of the proteolytic digestion sites either included orexcluded.

In yet another embodiment, special attention is given to theimmunogenicity of the fusion protein by altering the junction betweenthe two naturally occurring proteins from which it is made; theresulting fusion protein may be less or more able to elicit an immuneresponse against itself, which could lengthen its persistence orcontribute to it immunological effectiveness.

A hybrid nucleotide sequence of no more than 1528 base pairs including asequence defining a structural gene expressing a conjoined single strandof a multimeric TNFSF-SPD fusion protein, said structural gene having anucleotide base sequence selected from members of the group consistingof SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5 is disclosed by thisinvention. In one embodiment, the DNA segment the structural gene has asequence expressing a single hybrid amino acid chain of TNFSF-SPD, thesegment having a first SPD nucleotide base sequence of SEQ ID NO 1, frombase 32 to base 799, and a second sequence, expressing a portion ofTNFSF stalk, selected from members of the group consisting of SEQ ID NO1, from base 800 to base 1444, SEQ ID NO 3, from base 800 to base 1528,and SEQ ID NO 5, from base 800 to base 1441.

In another embodiment, a recombinant DNA molecule has vector operativelylinked to an exogenous DNA segment defining a structural gene expressinga single amino acid chain of TNFSF-SPD. This structural gene has anucleotide base sequence selected from members of the group consistingof SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5, any functional equivalentsand modifications thereof There is also attached an appropriate promoterfor driving the expression of said structural gene in a compatible hostorganism. The organism can be E. coli, a yeast, a higher plant oranimal.

Yet another embodiment contemplated by the invention is multimericTNFSF-SPD fusion protein having a plurality of polypeptide trimers, afirst trimer consisting of peptide strands of members of the TNFsuperfamily (TNFSF) of ligands, and a second trimer strand from acollectin molecule, each first trimer conjoined to a second polypeptidetrimer strand from a collectin molecule, wherein said ligand strand issubstituted for native carbohydrate recognition domains (CRD) of thecollectin molecules. The conjoined collectin strands are covalentlybound in parallel to each other, forming a multimeric fusion proteincomprising a plurality of trimeric hybrid polypeptide strands radiatingfrom a covalently bound center hub of the molecule. The free end of eachtrimeric radiating strand has a TNFSF moiety attached. The TNFSF moietyis one selected from the group consisting of ligands LTA, TNF, LTB, andTNFSF4 to TNFSF 18 as shown in Table II, and their funtionalequivalents, and modifications thereof.

The invention also contemplates a method for preparing a CD40-SPDmultimeric fusion polypeptide, including the steps of initiating aculture, in a nutrient medium, of procaryotic or eucaryotic host cellstransformed with a recombinant DNA molecule including an expressionvector, appropriate for the cells, operatively linked to an exogenousDNA segment defining a structural gene for CD40-SPD ligand. Thestructural gene has a nucleotide base sequence of SEQ ID NO 1 from aboutbase 32 to about base 1444. Thereafter, the culture is maintained for atime period sufficient for the cells to express the multimeric molecule.

Also contemplated is a method of producing a secreted, very large,biologically active, multimeric tumor necrosis factor superfamily ligandfusion protein chimera that is highly immunogenic and not readilydiffusable. The steps for this method are as follows:

-   -   1. introducing into a host cell a first chimeric DNA construct        including a transcriptional promoter operatively linked to a        first secretory signal sequence, followed downstream by, and in        proper reading frame with a first DNA sequence encoding a        polypeptide chain of a first TNFSF ligand requiring        multimerization for biological activity. This sequence is joined        to a second DNA sequence encoding a collectin polypeptide at the        site where the collectin's CRD was purposefully removed.

2. introducing into the host cell, a second DNA construct including atranscriptional promoter operably linked to a second secretory signalsequence followed downstream by, and in proper reading frame with, athird DNA sequence encoding a second polypeptide chain of a second TNFSFligand joined to a fourth DNA sequence encoding a collectin polypeptide,wherein the collectin's CRD was purposefully removed, and then,

-   -   3. growing the host cell in an appropriate growth medium under        physiological conditions to allow the secretion of a large        multimerized polypeptide fusion protein, wherein the first        polypeptide chain of a TNFSF-SPD protein is bound by parallel        bonding of the respective collectin domain trimer to the second        polypeptide chain of a different TNFSF-SPD polypeptide trimer,        and wherein the multimerized polypeptide fusion protein exhibits        biological activity characteristic of both membrane-attached        TNFSFs, and    -   4. isolating the biologically active, multimerized TNFSF-SPD        polypeptide fusion from said host cell. The chimeric reactant        compounds are humanized to guard against destruction by a        potential human recipient's immune system.

A final method of preparing a multimeric TNFSF-SPD ligand fusion proteincontemplated requires a) preparing a first DNA segment coding for astrand of an exposed extracellular portion of TNFSF; b) preparing asecond DNA segment coding for a collectin polypeptide strand, whereinthe collectin's CRD domain of the strand has been removed; c) conjoiningthe first and second DNAs in proper reading frame, thereby creating aTNFSF-collectin DNA construct; d) inserting the construct into anexpression vector system; e) introducing the vector system into anappropriate cell in culture under suitable conditions; f) harvesting andpurifying spent medium from the culture; and finally g) assaying forpresence of multimeric TNFSF-collectin fusion protein.

A method for stimulating the immune response in potentiallyimmonocompetent cells using multimeric TNFSF fusion proteins bycontacting the cells with the multimeric TNFSF fusion proteins, causingthe cells to proliferate, is also contemplated. The cells used may beresting B cells. There is also a method for increasing antigenicity ofcells by contacting the cells with the multimeric TNFSF fusion proteins.In this case, the cells may be tumor cells or HIV positive cells.

Other preferred embodiments contemplate the methods of preparationdescribed above, wherein the host transformed is either a prokaryote,such as E. coli, a eukaryote, for example yeast, such as S. cerevisiae.or a higher plant, such as alfalfa or tobacco.

Still further embodiments and advantages of the invention will becomeapparent to those skilled in the art upon reading the entire disclosurecontained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of the CD40L-SPD fusion protein. The extracellularportion of the CD40L homotrimer, including its membrane-proximal stalk,was fused to the body of SPD. The N-terminus of SPD contains twocysteines which link the homopolymer together by disulfide bonds forminga hub. The trimeric collagenous stalk extend from the hub as a cruciatestructure and end in a spontaneously trimerizing neck region. The aminoacid domains in a single chain of the CD40L-SPD are shown at the top. Atthe bottom is the tetrameric (four CD40L trimers) which is expected toform. In addition, the hub region of SPD can participate in stacking upto 8 or more cruciate forms into higher order aggregates.

FIG. 2. Ion-exchange chromatography of murine CD40L-SPD. CHO cellsexpressing murine CD40L-SPD were grown in serum-free media, concentratedusing a 100 kDa cutoff ultrafiltration membrane, and diafiltered into 50mM bicine, pH 9.0, 1 mM EDTA. Using an FPLC system, the protein from 400mL of media was applied to a Fractogel SO₃ 650M column and eluted with alinear salt gradient. 3 mL samples were collected. Shown are curves forprotein concentration (OD₂₈₀), conductivity as % 1 M NaCl in the buffer,and ELISA-detectable CD40L-SPD assayed at 1:100 dilution.

FIG. 3. Size fractionation of murine CD40L-SPD by ultrafiltration.CD40L-SPD is a 471 amino acid protein with a predicted molecular weightof 49,012 for each of the twelve component chains in the dodecamer(composed of four trimeric subunits). This does not include addedcarbohydrates. Therefore, the full dodecamer will have a molecularweight in excess of 600,000. However, from the literature on recombinantsurfactant protein D made in CHO cells, it appears that some of theproduct will be in the form of trimers that are not part of acruciate-formed dodecamer. To determine what percentage of CD40L-SPD wasproduced in a multimeric form, supernatant from the transfected CHOcells were passed through filters of different porosities (rated fortheir ability to retard globular proteins). An ELISA was used to detectthe amount of CD40L-SPD (measured at multiple dilutions) that passedthrough the filter. As shown, about 96% of the protein is retained by a300,000 kDa cut-off filter. This indicates that most of the protein isin the dodecameric form. In addition, the cruciate dodecamers ofsurfactant protein D can also stack on top of each other into evenhigher molecular weight forms. This is the likely explanation for thesmall fraction of CD40L-SPD that is retained by the 1,000 kDa cut-offfilter.

FIG. 4. Activation of human B cells by human CD40L-SPD. Conditionedmedia from CHO cells expressing human CD40L-SPD was added to human Bcells along with IL-4. In the left panel, the cells were stained withCyChrome-labeled anti-CD19 to identify B cells and PE-labeled anti-CD3to identify T cells. As shown, most of the cells proliferating in theculture were CD19+CD3-B cells. In the right panel, the cells werestained with CyChrome-labeled anti-CD19 to identify B cells andPE-labeled anti-CD80 (B7-1) to identify this co-stimulatory molecule. Asshown, almost all of the B cells were induced by CD40L-SPD to expressCD80.

FIG. 5. Activation of murine B cells by murine CD40L-SPD. MurineCD40L-SPD was added to resting murine splenic B cells for a two dayculture period. For the final 4 hours, the cultures were pulsed with³H-thymidine, following which the cells were harvested and DNA synthesiswas measured by scintillation counting. As shown, CD40L-SPD is nearly aseffective as anti-IgM in promoting the proliferation of resting B cells.

FIG. 6. CD40L-SPD stimulation of macrophage chemokine production.Conditioned media from CHO cells expressing human CD40L-SPD, an inactivemutant of human CD40L-SPD (T147N-CD40L-SPD), or murine CD40L-SPD(mCD40L-SPD) were added to cultures of human monocyte-derivedmacrophages. As a negative control, this media was heat-inactivated at60° C. for 30 minutes. Also shown is a form of soluble CD40L (sCD40L)consisting of 149 amino acids from the extracellular domain of humanCD40L (Peprotech) added at 1 ?g/mL. 24 hours later, supernatants werecollected and assay for MIP-1? by ELISA (R & D Systems). The weakactivity of soluble single-trimer CD40L (sCD40L) is apparent. Incontrast, native human and murine CD40L-SPD strongly activated themacrophages to produce MIP-1?. In contrast, heat-inactivated CD40L-SPDwas inactive. As expected, the inactive mutant, T147N-CD40L-SPD, alsofailed to stimulate macrophages, demonstrating that the CD40L portionand not the SPD portion of the protein was responsible for stimulatingthe macrophages.

FIG. 7. Expression of RANKL/TRANCE-SPD production from CHO cellsdetected by ELISA. Antibodies against RANKL/TRANCE were used toconstruct an ELISA capable of detecting the RANKL/TRANCE protein. Asshown, there was no background with the media control. Using a fusionprotein between CD70 (CD27L or TNFSF7) and SPD, there was also nosignal, indicating the specificity of the ELISA. However, using CHOcells transfected with an expression plasmid for CD70-SPD,immunoreactive secreted protein was clearly detectable. Thisdemonstrates the generalizability of the method for expressing TNFSFmembers as fusion proteins with collectins such as SPD.

DESCRIPTION OF THE PREFERRED EMBODIMENT

1. Definition of Terms

Multimeric: As used herein the term multimeric refers to a multimer of apolypeptide that is itself a trimer (i.e., a plurality of trimers).

Functional Equivalent: Herein refers to a sequence of a peptide orpolypeptide that has substantial structural similarity and functionalsimilarity to another such sequence.

Modifications: Herein refers to point changes involving single aminoacids, wherein the functionality is altered, without appreciablyaltering the primary sequence or primary structure of a peptide orpolypeptide.

Amino Acid: All amino acid residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature, J.Biol. Chem. 243: 3557-59, (1969), abbreviations for amino acid residuesare as shown in the following Table of Correspondence: TABLE OFCORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr L-tyrosine GGly glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine SSer L-serine L lie L-isoleucine L Leu L-leucine T Thr L-threonine V ValL-valine P Pro L-proline K Lys L-lysine H His L-histidine Q GinL-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine DAsp L-aspartic acid N Asn L-asparagin C Cys L-cysteine

It should be noted that all amino acid residue sequences are representedherein by formulae whose left to right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a bond to a radical such as Hand OH (hydrogen and hydroxyl) at the amino- and carboxy-termini,respectively, or a further sequence of one or more amino acid residuesup to a total of about fifty residues in the polypeptide chain.

Base Pair (bp): A partnership of adenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a doable stranded DNA molecule.

Constitutive promoter: A promoter where the rate of RNA polymerasebinding and initiation is approximately constant and relativelyindependent of external stimuli. Examples of constitutive promotersinclude the cauliflower mosaic virus 35S and 19S promoters described byPoszkowski et al., EMBO J, 3: 2719 (1989) and Odell et al., Nature, 313:810 (1985).

DNA: Desoxyribonucleic Acid.

Enzyme: A protein, polypeptide, peptide RNA molecule, or multimericprotein capable of accelerating or producing by catalytic action somechange in a substrate for which it is often specific.

Expression vector: A DNA sequence that forms control elements thatregulate expression of structural genes when operatively linked to thosegenes.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a structural gene to producea polypeptide.

Insert: A DNA sequence foreign to the rDNA, consisting of a structuralgene and optionally additional DNA sequences.

Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety(pentose), a phosphate, and a nitrogenous heterocyclic base. The base islinked to the sugar moiety via the glycosidic carbon (1′ carbon of thepentose) and that combination of base and sugar is a nucleoside. Whenthe nucleoside contains a phosphate group bonded to the 3′ or 5′position of the pentose it is referred to as a nucleotide.

Operatively linked or inserted: A structural gene is covalently bondedin correct reading frame to another DNA (or RNA as appropriate) segment,such as to an expression vector so that the structural gene is under thecontrol of the expression vector.

Polypeptide and peptide: A linear series of amino acid residuesconnected one to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a gene and to which RNApolymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Inducible promoter: A promoter where the rate of RNA polymerase bindingand initiation is modulated by external stimuli. Such stimuli includelight, heat, anaerobic stress, alteration in nutrient conditions,presence or absence of a metabolite, presence of a ligand, microbialattack, wounding and the like.

Spatially regulated promoter: A promoter where the rate of RNApolymerase binding and initiation is modulated in a specific structureof the organism such as the leaf, stem or root. Examples of spatiallyregulated promoters are given in Chua et al., Science, 244: 174-181(1989).

Spatiotemporally regulated promoter: A promoter where the rate of RNApolymerase binding and initiation is modulated in a specific structureof the organism at a specific time during development. A typicalspatiotemporally regulated promoter is the EPSP synthase-35S promoterdescribed by Chua et al., Science, 244: 174-181 (1989).

Temporally regulated promoter: A promoter where the rate of RNApolymerase binding and initiation is modulated at a specific time duringdevelopment. Examples of temporally regulated promoters are given inChua et al., Science, 244: 174-181 (1989).

Protein: A linear series of greater than about 50 amino acid residuesconnected one to the other as in a polypeptide.

Recombinant DNA molecule: A hybrid DNA sequence comprising at least twonucleotide sequences not normally found together in nature.

RNA: Ribonucleic Acid.

Selective Genetic marker: A DNA sequence coding for a phenotypical traitby means of which transformed cells can be selected from untransformedcells.

Structural gene: A DNA sequence that is expressed as a polypeptide,i.e., an amino acid residue sequence.

Synthetic promoter: A promoter that was chemically synthesized ratherthan biologically derived. Usually synthetic promoters incorporatesequence changes that optimize the efficiency of RNA polymeraseinitiation.

2. Introduction

This invention discloses the production of TNFSF proteins as multimeric(i.e., many trimers) ligands fused onto a trimeric, branched proteinbackbone. Collectin molecules are ideal for this purpose because theyare formed from many trimeric, collagenous arms linked to a central hubby disulfide bonds. Of the collectins, pulmonary surfactant protein D(SPD) was chosen initially because it is a homopolymer encoded by asingle gene, unlike C1q and surfactant protein A, which are composed oftwo different protein subunits. In addition, recombinant SPD has beensuccessfully expressed in vitro in reasonable yield [Crouch, 1994], anda peptide containing the “neck” region of SPD was shown to spontaneouslytrimerize in solution [Hoppe, 1994]. Consequently, extracellular domainsof human and murine CD40L were substituted for the carbohydraterecognition domain of pulmonary surfactant D (SPD) to create afour-armed molecule (three peptide chains per arm) with CD40L at the endof each arm. This molecule is named CD40L-SPD. In addition, because SPDtends to stack into higher order aggregates with up to 8 moleculesassociated at the hub [Crouch], even greater degree of multimerizationcan occur [Lu, 1993]. CD40L-SPD therefore mimics the expression of CD40Lby an activated T cell in that it presents a multivalent complex similarto membrane-bound CD40L. While remaining soluble, CD40L-SPD equalsmembrane CD40L in its range of activities.

3. Construction of Expression Plasmids for CD40L-SPD.

cDNAs of exposed human and murine CD40L, removed from cell membranes,were cloned by PCR by well-known methods. Murine surfactant protein Dwas cloned by hemi-nested PCR from murine lung mRNA (Clonetech). cDNAwas prepared using Superscript II reverse transcriptase (LifeTechnologies, Gaithersburg, Md.) and random hexamers as primers. PCRprimer sequences were as follows (the underlined bases indicaterestriction endonuclease sites for cloning into the vector): mSPD5:5′-CTGACATGCTGCCCTTTCTCTCCATGC-3′ mSPD3ext:5′-GGAGGCCAGCTGTCCTCCAGCCTGC-3′ mSPD5: 5′GGGG′CTAGCGAATTCCACCAGGAAGCAATCTGACATGC TGCCCTTTCTCTCCATGC-3′ CD40L/5′-CTATClTGTCCAACClTCTATG/GCCATCAGGGAACAA SPD3: TGCAGCTTTC-3′ SPD/5′-AAAGCTGCATTGTTCCCTGATGGC/CATAGAAGGTTGG CD40L5: ACAAGATAGAAG-3′CD40L3: 5′-GGGCTCGAGGTACCAGTTCTACATGCCTTGGAGTGTAT AAT-3′ SPD/5′-GAAAGCTGCATTGTTCCCTGATGGC/CATAGAAGATTG mCD40L5: GATAAGGTCGAAG-3′mCD40L/ 5′-CTTCGACCTTATCCAATCTTCTATG/GCCATCAGGGAA SPD3: CAATGCAGCTTTC-3′mCD40L3: 5′-GGGGGGTACCCTGCTGCAGCCTAGGACAGCGCAC-3′

Because the murine SPD sequence of the 5′ untranslated region containingthe ribosomal binding site was unknown when this work was started[Motwani, 1995], a primer (rmSPD5) was designed based on the availablerat sequence [Shimizu, 1992] which extended the 5′ end with rat sequence(shown in bold) along with an added Nhe I site (underlined).

4. Creation of the CD40L-SPD Fusions.

To create the CD40L-SPD fusions, overlap PCR was used. Murine SPD wasamplified by nested PCR using mSPD5 and mSPD3ext for the first round of30 cycles. The product was diluted 1:1,000 and 1 □L was amplified foranother 30 cycles using rmSPD5 and CD40L/SPD3, where the 3′ half ofCD40L/SPD3 is a reverse primer for SPD C-terminal to the neck region(deleting the CRD) and the 5′ half of CD40L/SPD3 contains bases from theN-terminus of the extracellular portion of CD40L (immediately adjacentto the transmembrane region). Similarly, the CD40L plasmid was amplifiedwith SPD/CD40L5 and CD40L3, which contains a Kpn I site (underlined).All of these PCRs were performed with Pfu cloned polymerase(Stratagene,) using hot start (Ampliwax, Perkin-Elmer) and thethermocycling program: 94° C. for 2.5 min; then 30 cycles of 94° C. for10 sec, 43° C. for 30 sec, and 75° C. for 7 min.

To form the chimeric construct, 1 μL of a 1:1,000 dilution ofgel-purified products from the above reactions was combined andamplified with rmSPD5 and CD40L3. Because Pfu polymerase did notconsistently yield the expected 1.62 kb overlap product, AccuTaq LA DNApolymerase (Sigma) was used for this PCR, using the thermocyclingprogram: 94° C. for 2.5 min; then 30 cycles of 98° C. for 20 sec, 43° C.for 30 sec, and 68° C. for 10 min. The resulting product was digestedwith Nhe I and Kpn I, gel-purified, and ligated into the Nhe I and Kpn Isites in the expression plasmid, pcDNA3.1(+) (Invitrogen, Carlsbad,Calif.). DH5 E. coli were transformed with the construct and plasmid DNAwas purified either by double banding in ethidium bromide-CsCl gradientsor by anion exchange resin (QIAgen). To form the T147N-CD40L-SPDconstruct, the same approach was used except that the CD40L codingregion was taken from the expression plasmid for T147N-CD40L[Kornbluth]. The amino acid sequence at the junction between SPD andCD40L is . . . KAALFPDG/HRRLDKIE . . . , where the C-terminal portionbegins the sequence for CD40L. To form mCD40L-SPD, a similar approachwas taken except that primers SPD/mCD40L5, mCD40L/SPD3, and mCD40L3 wereused for amplifications involving murine CD40L sequences. The amino acidsequence at the junction between SPD and murine CD40L is . . .KAALFPDG/HRRLDKVE . . . , where the C-terminal portion begins thesequence for murine CD40L. Both DNA strands of each construct weresequenced to confirm that the constructs were correct. In otherexperiments, an entirely humanized construct, consisting of human CD40Lfused to human SPD, was constructed (data not shown).

5. Construction of Expression Plasmid for Murine RANKL/TRANCE (TNFSF11).

Spleen cells from C3H/HeJ mice were stimulated with 5 μg/ml concanavalinA and 10 ng/ml IL-2 (Sigma) for 8 hours (31). mRNA was isolated usingthe Micro FastTrack kit (Invitrogen). cDNA was prepared usingSuperscript II reverse transcriptase (Life Technologies) and randomhexamers as primers. PCR primer sequences were as follows (where theunderlined bases indicate restriction endonuclease sites for cloninginto the vector): 5mRANKL-ext: 5′-CATGTTCCTGGCCCTCCTC-3′ 3mRANKL-ext:5′-GTACAGGCTCAAGAGAGAGGGC-3′ 5mRANKL-int:5′-ATACTCGAGCGCAGATGGATCCTAAC-3′ 3mRANKL-int:5′-GGGGTTTAGCGGCCGCTAATGTTCCACGAAATGAGTTC-3

The extracellular portion of RANKL/TRANCE was cloned by nested PCR. Inthe first round of PCR, 5mRANKL-ext and 3mRANKL-ext were used with Pfucloned polymerase (Stragene) using the thermocycling program: 94° C. for2.5 min; then 30 cycles of 94° C. for 10 sec, 50° C. for 30 sec, and 75°C. for 2 min. The product was diluted 1:1,000 and 1 μL was amplified foranother 30 cycles using 5mRANKL-int and 3mRANK-int, which contain an XhoI site and a Not I site respectively. The resulting product was digestedwith Xho I, blunt-ended with T4 DNA polymerase, then digested with Not Iand gel-purified. The CD40L-SPD expression piasmid described above wasdigested with Msc I and Not I and gel purified. Then the RANKL/TRANCEsequence was ligated into this vector in frame with the SPD codingsequence. The amino acid sequence at the junction between SPD andRANKL/TRANCE is . . . KAALFPDG/RAQMDPNR . . . , where the N-terminalportion is from SPD and the C-terminal portion is the extracellularsequence of RANKL/TRANCE. Both DNA strands of each construct weresequenced to confirm that the constructs were correct.

6. Stable Transfection of DHFR-Deficient CHO Cells and Amplification.

DG44 (a line of CH0-K1 cells deficient in dihydrofolate reductase(DHFR)) (32) and pCHIP (a plasmid containing the hamster DHFR minigene)(33) were gifts from Dr. Lawrence Chasin, Columbia University, New York,N.Y. DG44 cells were cultured in α-MEM consisting of ribo- anddeoxynucleoside-free α-MEM (BioWhittaker, Walkersville, Md.)supplemented with 200 μM L-glutamine, 10% fetal bovine serum (FBS) and10 μg/ml each of adenosine, deoxyadenosine, and thymidine (Sigma). Allcell cultures described were negative in a mycoplasma rRNA assay(Gen-Probe, San Diego). DG44 cells in six-well plates were transfectedby the method of Okayama and Chen ((34) with 10 μg of expression plasmidand 0.05 μg of pCHIP (200:1 ratio). After two days, the transfected DG44were trypsinized and transferred to 100 mm plates. At this point, themedia was switched to α-MEM which differs from α-MEM in that dialyzedFBS (HyClone Systems, Logan, Utah) was used and no nucleosidesupplements were added. Only cells containing the DHFR minigene wereable to grow in α-MEM, and colonies were selected after 10 days, clonedusing cloning rings, and transferred to 12.5 cm² flasks. Clones wereselected for expansion using an ELISA to screen for the production ofeither raurine or human CD40L (see below). Using the method described byKingston et al. (35), escalating doses of methotrexate were used toamplify the transfected genes over a period of 6-14 months until thecells grew well in 80 μM methotrexate. Each expressing clone wasre-cloned once or twice more in order to select the highest expressingcells.

7. Preparation of Human and Murine CD40L-SPD in Serum-Free Media.

Selected clones were adapted for growth in nucleoside-free UltraCHOmedia (BioWhittaker) supplemented with 50-100 μg/mL ascorbic acid and 50μM methotrexate (Sigma). The non-adherent population was further adaptedfor suspension growth in roller bottles. In some experiments, the cellswere adapted from α-MEM to CHO-S-SFM II media (Life Technologies)supplemented with ascorbic acid and 50 μg/mL L-proline.

8. ELTSA Assay for Human and Murine CD40L-SPD.

To assay for correctly folded CD40L, wells of a MaxiSorb 96-well plate(Nunc) were coated overnight at 4° C. with 50 ?L ofcarbonate-bicarbonate, pH 9.40 buffer containing 0.5 μg/mL 24-31anti-human CD40L MAb (Ancell) or MR1 anti-murine MAb (Bioexpress,Lebanon, N.H.). Wells were blocked with 3% bovine serum albumin (BSA) inPBS. 100 μL samples were added to the wells either neat or diluted in adilution buffer consisting of 1% BSA, 0.9% NaCl, 50 mM Tris pH 7.40, and0.1% peroxide-free Tween 20 (Sigma). After shaking for 2 h at 600 RPM, aplate washer was used to wash the plate four times with 0.9% NaCl, 50 mMTris pH 7.40, and 0.1% peroxide-free Tween 20. Then, 100 μL of diluentbuffer containing 1 μg/mL biotinylated 24-31 anti-human CD40L Mab(Ancell) or MR1 anti-murine CD40L Mab (Pharmingen, San Diego, Calif.)was added to each well and again shaken for 2 h. Following another fourwashes, 100 μL of diluent buffer containing 1 μg/mL ofstreptavidin-alkaline phosphatase (Jackson) was added to each well andthe plate was shaken for 1 hour. Lastly, after another four washes,color was developed for 10-20 min using 100 μL/well of BluePhos(Kierkegaard & Perry), stop solution was added, and the wells were readat 650 μm in a plate reader.

9. Purification of Human and Murine CD40L-SPD.

Conditioned UltraCHO media was filtered using a 0.2μ PES filter unit(Nalgene) and stored at 4° C. for up to 3 months. A preliminary sizefractionation was performed by ultrafiltration through a 100 kDa-cutoff76 mm membrane (YM-100, Millipore) in a 400 mL stirred cell at 10lbs/sq. inch pressure of argon. Media was concentrated to about 10 mL,diluted to 100 mL with buffer, and again concentrated to 10 mL for atotal of 3 cycles of ultrafiltration and buffer exchange. Buffer was 50mM Bicine (Calbiochem), adjusted to pH 9.0 with NaOH (about 32 mM Na),and 1 mM EDTA to prevent the activity of any metalloproteinase. UsingFPLC equipment (Amersham-Pharmacia), the concentrate was filteredthrough a 0.45μ filter, placed into a 10 mL superloop, applied to a10×30 mm column (HR10/30, Amersham-Pharmacia) packed with Fractogel SO₃650M (EM Biosciences), and eluted at 0.5 mL/min at 4° C. with a lineargradient of 0-500 mM NaCl in buffer. As described by the manufacturer,the resolution of proteins on Fractogel SO₃ is enhanced by using a long,thin column geometry. Fractions were collected and screened for human ormurine CD40L by ELISA. Positive fractions were pooled, concentrated byultrafiltration (CentriPrep-30, Millipore), filtered through a 0.45μfilter, and applied to a Superose 6 column (Amersham-Pharmacia) inphosphate-buffered saline.

10. Murine B Cell Cultures.

C3H/HeJ mice were euthanized by CO₂ inhalation under a protocol approvedby the Animal Subjects Committee of the San Diego VA Healthcare System.Splenocytes were isolated by centrifugation over Lympholyte-M (AccurateChemical & Scientific Corp., Westbury, N.Y.) and B cells were isolatedby negative selection using anti-CD43 immunomagnetic beads (MiltenyiBiotec Inc., Auburn, Calif.). The resting B cells were suspended inDulbecco's MEM with 10% FBS at a concentration of 1×10⁶/mL, and 100 μLwas added to the wells of 96-well flat-bottomed plates. 100 μL ofdilutions of murine CD40L-SPD in media or media alone were added to thewells, which were incubated in 8.5% CO₂ at 37° C. for 48 hours. Then,0.5 μCi/well of ³H-thymidine was added to each well, and the cells werecollected 4 h later onto glass fiber filters using an automated cellharvester. A scintillation counter was used to determine theincorporated radioactivity.

11. Human B Cell Cultures.

Venous blood from consenting subjects was used as a source of human Bcells under a protocol approved by the UCSD Institutional Review Board.Blood was collected into syringes containing 5 U/mL heparin andperipheral blood mononuclear cells (PBMC) were isolated bycentrifugation over Ficoll-hypaque. The cells were suspended at 2×10⁵/mLin RPMI 1640 containing 200 μM L-glutamine, 10% FBS, 0.832 μMcyclosporin A (Sigma), and 25 ng/mL human IL-4 (R & D Systems) andincubated in 5% CO₂ at 37° C. as described by Schultze et al. (36). Atintervals, the cells were stained with CyChrome-conjugated anti-CD19 andPE-conjugated anti-CD80 (B7-1) monoclonal antibodies (Pharmingen) andanalyzed by flow cytometry.

12. Human Monocyte-Derived Macrophage and Dendritic Cell Cultures.

As previously described [Kornbluth], monocytes were isolated from PBMCby adherence to fibronectin-coated plates, plated into 48-well plates,and then cultured in RPMI1640 containing 200 ?M L-glutamine and 10%autologous serum for 7-10 days. Monolayers of the matured cells (about2×10⁵/well), termed monocyte-derived macrophages or MDM, were thenwashed in media and cultured in 1 mL/well RPMI1640 containing 200 ?ML-glutamine and 10% heat-inactivated FBS. Alternatively, dendritic cells(DC) were formed from monocytes by adding GM-CSF and IL-4 to the culturemedia, and the resulting DC were used 6 days later. Preparations ofCD40L-SPD were added to the wells as indicated. As a positive control,100 ng/mL bacterial lupopolysaccharide (LPS) from E. coli 0111:B4(Calbiochem) was added. Supernatants were collected 24 h later andanalyzed for cytokine content using ELISA (R & D Systems).

EXAMPLE 1 Design Principles in Constructing Collectin-TNFSF MemberFusion Proteins

To express CD40L and other TNFSF members as stable, multimeric proteins,the coding region of the extracellular, C-terminal portion of CD40L wasjoined in-frame to the collectin, surfactant protein D (SPD). TheN-terminus of SPD contains two cysteines which form the disulfide bondsnecessary for the 4-armed cruciate structure of the overall molecule[Brown-Augsburger, 1996 #506]. C-terminal to these cysteines in SPD is along triple-helical collagenous “stalk” which ends in the “neck” regionthat promotes the trimerization of each arm of the structure.Immediately after this neck region, the coding sequence for theextracellular portion of CD40L was added, in place of the carbohydraterecognition domain (CRD) of SPD. The collectins were chosen as theframework for the multimeric construct because of their multi-subunitstructure and the trimeric nature of their stalk regions.Appropriateness of replacing the CRD of a collectin with theextracellular region of a TNFSF member is further supported bystructural studies of the two protein families. An analysis of the CRDcrystal structure of another collectin, ACRP30, indicated that it wasstructurally superimposable upon the crystal structures of theextracellular regions of CD40L, TNF, and Fas [Shapiro, 1998]. Thesuccessful expression of the collectin-TNFSF fusion protein, CD40L-SPD,indicates that other TNFSF members (Table I) could be conjoined to SPDin a similar manner and that other collectins besides SPD (Table II)could be used as a protein framework instead of SPD. Because thesemolecules are formed entirely from naturally occurring proteins, theproduction of an immune response (e.g., antibodies) to these fusionproteins is minimized. By deleting portions of the stalk region of theTNFSF proteins, additional constructs can be made which may be even lessimmunogenic.

EXAMPLE 2 Expression of Human and Murine CD40L-SPD in CHO Cells

The coding regions for the extracellular portion of human CD40L, humanT147N-CD40L, an inactive mutant of CD40L, or murine CD40L were joined tothe neck region of murine SPD, replacing the SPD CRD (FIG. 1). ACMV-driven expression plasmid for the construct was co-transfected witha DHFR minigene into DNFR-deficient CHO cells. Following selection innucleoside-free media, expressing CHO clones were amplified by culturein ascending doses of methotrexate. The resulting clones produced about1-10 μg/mL of the fusion protein over a 3 day period in media containingFBS.

Clones were adapted for growth as suspension cells in two types ofsertim-firee media. Murine CHO-SPD produced in UltraCHO (BioWhittaker)was largely retained (about 60% as determined by ELISA) by a 1,000 kDacutoff ultrafiltration membrane (Pall Corp., Port Washington, N.Y.),consistent with a large multimeric complex formed by the stacking of theSPD portion of the molecule. However, in CHO-S-SFM II (LifeTechnologies), nearly all ELISA-detectable murine CHO-SPD passed througha 100 kDa cutoff ultrafiltration membrane (Millipore), suggesting thatthe protein was either folding incorrectly in this media or was beingdegraded by proteolysis. Consequently, the purification method wasoptimized for the spent UltraCHO media.

EXAMPLE 3 Purification of Human and Murine CD40L-SPD

Purification procedures were developed for murine CD40L-SPD, but thesame methods could be applied to human CD40L-SPD with minormodifications. Murine CD40L-SPD has a predicted m.w. of 49 kDa perchain, or about 600 kDa per 12-chain, cruciate molecule, the amino acidsequence predicts a pI of 9.10. Accordingly, conditioned media wasconcentrated by ultrafiltration through a 100 kDa cutoff filter, whichalso fractionates the sample on a size basis. After diafiltration into50 mM bicine, pH 9.00 (also containing 1 mM EDTA added to inhibitmetalloproteinases), the sample was applied to a variety of cationicexchange resins. Using Source 30S (Amersham-Pharmacia), most of theELISA-detectable protein did not bind and was recovered in theflow-through. However, as reported by Morris et al. {Morris}, FractogelSO₃650M retained the protein. The retention by this tentacular resin andnot by Source 30S suggests binding to positively charged residues thatare not on the protein surface. Using a linear NaCl gradient,ELISA-detectable protein elutes at between 0.15-0.30 M NaCl under theseconditions (FIG. 2). In selected experiments, the protein was furtherpurified using a Superose 6 sizing column. Most of the ELISA-detectableprotein eluted in the excluded volume, indicating an apparent m.w. ofgreater than 1,000 kDa (FIG. 3).

EXAMPLE 4 Activity of CD40L-SPD on Human B Cells

Schultze et al. described a system using CD40L-expressing cells plusIL-4 and cyclosporin A (to inhibit T cell growth) as a means to growvery large numbers of B cells from a small sample of blood. BecauseCD40L activates these B cells to express high levels of B7 molecules(CD80 and CD86), the proliferating B cells were effective in presentingpeptide antigens and rival non-dividing dendritic cells asantigen-presenting cells (APCs) (36). To determine if the CD40L-SPDfusion protein could replace CD40L-expressing cells in this system, PBMCwere cultured with CD40L-SPD in addition to IL-4 and cyclosporin A.Under these conditions the cells grew to saturation density every threedays. After three weeks, the cultures were almost entirely CD19+B cellswhich express high levels of CD80 (FIG. 4). This indicates thatCD40L-SPD can be used in ex vivo systems where a soluble yet effectiveform of CD40L is needed to stimulate cells for immunotherapeuticapplications.

EXAMPLE 5 Activity of CD40L-SPD on Murine B Cells

Resting murine B cells are particularly difficult to stimulate with mostsoluble forms of CD40L. Even with murine CD40L-CD8 fusion proteins, itis necessary to crosslink the protein with antibodies against CD8 inorder to achieve maximal proliferation in culture [Klauss, 1999].Accordingly, resting murine B cells were negatively selected withimmunomagnetic beads. As shown in FIG. 5, murine CD40L-SPD was aseffective as anti-IgM antibody in driving B cells to proliferate. Thisindicates that CD40L-SPD can mimic the multivalent interactions thatoccur when a responding cell comes in contact with CD40L-bearingactivating cells.

EXAMPLE 6 Activity of CD40L-SPD on Human Macrophages and Dendritic Cells

CD40L is a powerful stimulant for macrophages (reviewed in (28)) anddendritic cells (40). Accordingly, preparations of CD40L-SPD were addedto monocyte-derived macrophages and the production of MIP-1

was used as a measure of stimulation. As shown in FIG. 6, both human andmurine CD40L-SPD were able to stimulate macrophages, whereas theT147N-CD40L-SPD mutant was inactive as expected.

Discussion

These examples define a new method of producing multimeric (i.e., manytrimers) of CD40L as a fusion protein with SPD. Also prepared andexpressed were similar fusion proteins between raurine RANKL/TRANCE(TNFSF 11) or murine CD27L/CD70 (TNFSF7) joined to murine SPD (data notshown). This suggests that virtually all TNFSF members could besuccessfully produced as fusion proteins with SPD. Furthermore, it isalso likely that other collectins besides SPD could be used in thesefusions, given the strong structural homologies between the CRDs of thecollectins and the extracellular domains of TNFSF members [Shapiro]which can be substituted for these CRDs. Given the 17 known TNFSFmembers and 9 known collecting, at least 153 fusion protein combinationsare possible.

SPD was selected for initially because it is a soluble homopolymer.Other collecting, such as surfactant protein A, have strong bindingaffinities to lipids and specific cell receptors. Although removal ofthe CRD abrogates much of this binding, it may be partially mediated bythe neck region sequence, which the fusion proteins retain. Accordingly,it would be expected that collectins other than SPD might conferdifferent cell-binding and pharmacokinetic behaviors upon a fusionprotein. For example, macrophages are known to take up and degrade wholeSPD [Dong, 1998]. If a fusion protein other than SPD were used, thedisposition of the fusion protein in vivo might be altered.Additionally, metalloproteinases are known to degrade the collectin,C1q, so that a fusion with C1q may alter the degradation of the fusionprotein. For example, because CD40L activates macrophages and othercells to produce metalloproteinases, which could potentially degrade thecollagenous portion of SPD and other collecting. Cleavage of thecollagenous stalk would then be expected to release single-trimers ofCD40L, which could diffuse away from the original parent molecule, muchlike a slow-release formulation of a drug. Also, the membrane-proximalportion of CD40L has been retained in CD40L-SPD. This sequence alsocontains protease-susceptible amino acid sequences, which can beeliminated by mutagenesis to retard the cleavage of CD40L from thefusion protein. Mutations in such proteinase cleavage site(s) woulddelay such cleavage and favor the local persistence of the CD40Lstimulus.

CD40L-SPD is a large macromolecule (>1,000 kDa), and the otherTNFSF-collectin fusion proteins would be expected to be similarly large.For native SPD, the aggregates that spontaneously form measure 100 nm indiameter. When injected into tissue, this large a complex would beexpected to remain at the injection site for a prolonged period.Localization of the TNFSF-containing protein would also be expected toreduce any systemic toxicity caused by the release of freesingle-trimers into the circulation. For example, soluble CD40L in bloodhas been linked to disease activity in lupus, and this smaller moleculemay even cross the glomerulus to cause damage to renal tubules [Kato andKipps, J. Clin. Invest. November 1999]. On the other hand, because CD40Linduces the production of chemokines which attract immune cells[Kornbluth], T cells, monocytes, and dendritic cells would be expectedmigrate to the site where CD40L-SPD was injected. This might beadvantageous if CD40L-SPD were used as a vaccine adjuvant. In mice,soluble CD40L (sCD40LT) stimulates IgG1 production but not cytotoxic Tlymphocytes (CTLs) [Wong, 1999]. Interestingly, the same protein that isexpressed from an injected plasmid stimulates both a strong antibody andCTL response [Gurunathan, 1998]. In the latter case, the plasmid wouldbe expected to deliver a localized supply of CD40L, whereas the sCD40LTprotein is free to diffuse away. Support for the localized use of CD40Lin an adjuvant formulation is provided by a study using a plasmidexpressing full-length membrane CD40L, which was very effective instimulating both humoral and CTL immune responses [Mendoza, 1997].Similarly, injection of adenovirus expressing membrane CD40L has potentantitumor activity in mice [Kikuchi, 1999]. Similar considerations wouldlikely apply to other fusion proteins between the TNFSF and collectins.

Finally, for immunostimulatory proteins, it is particularly importantthat the protein not be antigenic if repeated injections are needed. Forexample, vaccination with TNF-β modified by the addition of shortpeptide sequences was able to induce the production of disease-modifyinganti-TNF-μ autoantibodies [Dalum, 1999]. Because CD40L-SPD and otherTNFSF-collectin fusion proteins are formed from endogenous proteinsequences (with the possible exception of the peptide sequence at thejunction), the production of antibodies might not limit theeffectiveness of repeated injections.

In conclusion, fusions between TNFSF members and collectins offer anovel means of generating large protein complexes which can providelocalized stimulation at an injection site. Because of the multimericnature of the collectin backbone, such fusion proteins may mimic themultivalent ligand surface presented by the membrane forms of TNFSFmembers to TNFRSF-bearing responding cells. Moreover, by limitingsystemic toxicity while maintaining localized efficacy, such fusionproteins may have a role as vaccine adjuvants against infectious agentsand tumors. TABLE I Ligands of the TNF Superfamily* New Ligand SymbolOther Names Genbank ID LTA Lymphotoxin-, TNF-?, TNFSF1 X01393 TNF TNF-?,TNFSF2 X02910 LTB Lymphotoxin-, TNFSF3 L11016 TNFSF4 OX-40L D90224TNFSF5 CD40L, CD154, Gp39, T-BAM X67878 TNFSF6 FasL U11821 TNFSF7 CD27L,CD70 L08096 TNFSF8 CD30L L09753 TNFSF9 4-1BBL U03398 TNFSF10 TRAIL,Apo-2L U37518 TNFSF11 RANKL, TRANCE, OPGL, ODF AF013171 TNFSF12 TWEAK,Apo-3L AF030099 TNFSF13 APRIL NM_003808 TNFSF13B BAFF, THANK, BLYSAF136293 TNFSF14 LIGHT, HVEM-L AF036581 TNFSF15 VEGI AF039390 TNFSF16Unidentified TNFSF17 Unidentified TNFSF18 AITRL, GITRL AF125303*(as of Nov. 1, 1999) Known members of ligands in the TNF superfamily,taken from the Human Gene Nomenclature Committee athttp://222.gene.ucl.ac.uk/users/hester/tnftop.htm

TABLE II The Collectin Superfamily Clq Pulmonary surfactantMannose-binding protein, protein D MBL1 conglutinin Mannose-bindingprotein, collectin-43 MBL2 CL-L1 Pulmonary surfactant ACRP30 protein AHib27

All collectins are formed as multimers of trimeric subunits, eachcontaining a collagenous domain. The C-terminus of each collectincontains a CRD which binds carbohydrates and other ligands. Because ofthe tight similarities between the known CRD structures and theextracellular domains of TNFSF members, it is likely that the CRD of anycollectin could be replaced with the extracellular domain of any TNFSFmember in a structurally compatible manner.

While the present invention has now been described in terms of certainpreferred embodiments, and exemplified with respect thereto, one skilledin the art will readily appreciate that various modifications, changes,omissions and substitutions may be made without departing from thespirit thereof. It is intended, therefore, that the present invention belimited solely by the scope of the following claims.

1-15. (canceled)
 16. A method of treatment of a disorder or disease in asubject comprising administration of a multimeric polypeptide of trimerunits, each unit comprising: a collectin family scaffold operably linkedto an extracellular domain of a tumor necrosis factor superfamily(TNFSF) polypeptide to form a polypeptide trimer wherein the multimericpolypeptide is at least a dimer of trimer units.
 17. The method of claim16, wherein the disorder or disease is an inflammatory disorder,autoimmune disease, infectious disease, viral infectious disease or atumor.
 18. An isolated nucleic acid sequence encoding a multimericpolypeptide of claim
 16. 19. An expression vector containing a nucleicacid sequence according to claim
 18. 20. A pharmaceutical compositioncomprising a nucleic acid sequence of claim
 18. 21. A method of treatinga disorder or disease in a subject comprising administration of anucleic acid sequence of claim
 18. 22. The method of claim 21, whereinthe disorder or disease is an inflammatory disorder, autoimmune disease,infectious disease, viral infectious disease or a tumor.
 23. A host cellcontaining an expression vector according to claim
 19. 24. The host cellof claim 23, wherein the host cell is a prokaryote.
 25. A method forproducing a multimeric polypeptide of trimer units, each unitcomprising: a collectin family scaffold operably linked to anextracellular domain of a tumor necrosis factor superfamily (TNFSF)polypeptide to form a polypeptide trimer wherein the multimericpolypeptide is at least a dimer of trimer units, the method comprisingculturing a host cell of claim 23 under conditions to produce themultimeric polypeptide.
 26. A method of stimulating a biologicalresponse in a subject in need thereof, comprising administering to thesubject a composition comprising a multimeric polypeptide of trimerunits or a polynucleotide encoding the multimeric polypeptide, each unitcomprising: a collectin family scaffold operably linked to anextracellular domain of a tumor necrosis factor superfamily (TNFSF)polypeptide to form a polypeptide trimer wherein the multimericpolypeptide is at least a dimer of trimer units, thereby stimulating abiological response in the subject.
 27. The method of claim 26, whereinthe subject in need thereof has a tumor.
 28. The method of claim 26,wherein the subject in need thereof has HIV positive cells.
 29. A methodof stimulating angiogenesis in a subject in need thereof, comprisingadministering to the subject a composition comprising a multimericpolypeptide of trimer units or a polynucleotide encoding the multimericpolypeptide, each unit comprising: a collectin family scaffold operablylinked to an extracellular domain of a tumor necrosis factor superfamily(TNFSF) polypeptide to form a polypeptide trimer wherein the multimericpolypeptide is at least a dimer of trimer units, thereby stimulatingangiogenesis in the subject.
 30. The method of claim 29, wherein theTNFSF polypeptide is TWEAK.
 31. The method of claim 29, wherein thesubject has coronary artery disease.
 32. A method of inhibitingangiogenesis in a subject in need thereof, comprising administering tothe subject a composition comprising a multimeric polypeptide of trimerunits or a polynucleotide encoding the multimeric polypeptide, each unitcomprising: a collectin family scaffold operably linked to anextracellular domain of a tumor necrosis factor superfamily (TNFSF)polypeptide to form a polypeptide trimer wherein the multimericpolypeptide is at least a dimer of trimer units, thereby inhibitingangiogenesis in the subject.
 33. The method of claim 32, wherein theTNFSF polypeptide is VEGI.
 34. The method of claim 32, wherein thesubject has a tumor.
 35. The method of claim 32, wherein the subject hasretinal neovascularization.
 36. A method of promoting wound healing in asubject in need thereof, comprising administering to the subject acomposition comprising a multimeric polypeptide of trimer units or apolynucleotide encoding the multimeric polypeptide, each unitcomprising: a collectin family scaffold operably linked to anextracellular domain of a tumor necrosis factor superfamily (TNFSF)polypeptide to form a polypeptide trimer wherein the multimericpolypeptide is at least a dimer of trimer units, thereby inhibitingangiogenesis in the subject.
 37. The method of claim 36, wherein theTNFSF polypeptide is APRIL.
 38. The method of claim 36, wherein thesubject has a poorly healing wound.